4Beta-1&#34;-{2&#39;-substituted benzoyl) anilino] podophyllotoxin analogues useful as anticancer agents

ABSTRACT

The present invention provides a new class of compounds 4β-1″-[{2″-benzoyl substituted}anilino]podophyllotoxin exhibiting anti cancer activity and a process for preparing the same.

TECHNICAL FIELD

The present invention relates to a process for the synthesis of new4β-1″-[2″-(substituted benzoyl)anilino]podophyllotoxin analogues areuseful anticancer agents. The present invention particularly relates tothe synthesis of new class of 4β-O-benzoyl anilino congeners of thepodophyllotoxin as useful anticancer agents.

BACKGROUND ART

Etoposide and teniposide two synthetic podophyllotoxin derivatives whichare important drugs that currently being used in the treatment of smalllung cancer, testicular carcinoma, lymphoma, Kaposi's sarcoma. Theclinical efficacy and intriguing mechanism of etoposide has greatlystimulated interest in further studies on the modification of the C-4substution of this compound and for better antitumour activity (Jadine,I. In Anticancer Agents Based on Natural Products Models; Cassady, J.M., Dours, J., Eds.; Academic press: New York, 1980, p 319.; Levy, R.K.; Hall, I. H.; Lee, K. H. J. Pharm. Sci. 1983,72,1158.; Issell, B. F.;Muggia, F. M.; Carter, S. K. Etoposide [VP-16]Current Status and NewDevelopments; Academic Press New York, 1984.; Stio, H.; Yoshikawa, H.;Nishimura, Y.; Kondo, S.; Takeuchi, T.; Umezawa, H. Chem. Pharm. Bull.1986,34,3733. Satio, H.; Nishimura, Y.; Kondo, S.; Komuro, K.; Takeuchi,T.; Bull. Chem. Soc. Jpn.1988, 61, 2493). It has been well establishedthat the principal mechanism of the action for etoposide is by theinhibition of catalytic activity of DNA topoisomarase II and concurrentenzyme mediated production of lethal DNA strand breaks. Structureactivity relationship studies for the podophyllotoxin-derived compoundshave shown the trans C/D ring juncture is a essential for the antitumouractivity. A number of studies have been carried out on the structuralmodification of glycoside moiety by 4-alkylamino or 4-arylaminosubstituents have improved the inhibitory activity on human DNAtopoisomarase II as well as stronger activity in causing cellularprotein length DNA breakage (Lee. K. H.: Imakura. Y.: Haruna. M.; Beers,S. A.; Thurston, L. S.; Dai, H. J.; Chen, C. H.; Liu, S. Y.; Cheng, Y.C. J. Nat. Prod. 1989, 52, 606. Liu, S. Y.; Hawang, B. D.; Haruna, M.;Imakura, Y.; Lee, K. H.; Cheng, Y. C. Mol. Pharmcol. 1989, 36, 78. Lee,K, H.; Beers, S. A.; Mori, M.; Wang, Z. Q.; Kuo, Y. H.; Li, L.; Liu, S.Y.; cheng, Y. C.; J. Med. Chem. 1990, 33,1364). In the context a largenumber of 4β-aryl amino derivatives of 4′-O-demethyl epipodophyllotoxinbased compound have been synthesized and investigated for theirantitumour activity.

OBJECTIVES OF THE INVENTION

The main object of the invention is to provide the new4β-[2″-benzoylsubstituted]arylamino podophyllotoxin analogues useful asanticancer agents.

Another object of the present invention is to provide a process for thesynthesis of new 4β-1″-[{2″-benzoyl substituted}anilino]podophyllotoxinderivatives as useful anticancer agents, which obviates the draw backsas detailed above.

Another object of the present invention is to provide new andstereo-selective compounds of the podophyllotoxins and4′-O-demethylepipodophyllotoxin in good yields.

Still another object of the present invention is to provide the key stepfor the synthesis of these analogues by direct nucleophilic substitutionof the C-4β-bromo intermediate.

SUMMARY OF THE INVENTION

The above and other objective of the present invention are achieved byproviding the new class of C₄-62 -aryl substituted and N-linkedderivatives of podophyllotoxin and 4′-O-demethylepipodophyllotoxin,which have been synthesized as anti cancer agents.

Accordingly, the present invention provides new class of4β-1″-[(2″-benzoyl substituted}anilino]podophyllotoxin analogues havingthe structural formula (2). The present invention also provides aprocess for the preparation of new 4β-1″-[2″-(substitutedbenzoyl)anilino]podophyllotoxin analogues as useful anticancer agents.More particularly, it provides a process for the preparation of4β-1″-[2″-(substituted benzoyl)anilino] derivatives of podophyllotoxin.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 represents structure of podophyllotoxin, Etoposide andTeniposide.

FIG. 2 represents general formula of compounds of the class4β-1″-[{2″-benzoyl substituted}anilino]podophyllotoxin analogues.

FIG. 3 discloses the process for the synthesis of new podophyllotoxinanalogues as anticancer agents producing the novel and stereo-selectivederivatives of the podophylotoxin in good yields.

DETAILED DESCRIPTION

The process for the synthesis of new podophyllotoxin analogues asanticancer agents produces the novel and stereo-selective derivatives ofthe podophyllotoxin in good yields, where in the key step for thesynthesis of these analogues is by direct nucleophilic substitution ofC-4β-bromo intermediates, 4β-bromo-podophyllotoxin and4′-O-demethylepipodophyllotoxin, which have been reacted withsubstituted or unsubstituted 2-aminobenzophenones in a stereo-selectivemanner to afford the 4β-1″-[{2″-benzoylsubstituted}anilino]podophyllotoxin derivatives.

These 4-bromopodophyllotoxin intermediates have been prepared by thebromination of the related podophyllotoxin compounds as described in theliterature [Kuhn, M.; Keller-Juslen, C.; Van Wartburg, Helv. Chemica.Acta, 1969, 52, 944].

In an embodiment of the present invention, the naturally occurringpodophyllotoxin lignan was isolated from Podophyllum peltatum linnaeus.

In another embodiment of the present invention the synthesis of4β-intermediates have been carried out from bromination ofpodophyllotoxin and 4′-O-demethylepipodophyllotoxin.

In yet another embodiment of the present invention 1-2 eq. of differentunsubstituted and substituted benzophenone compounds have been used.

In still another embodiment of the present invention a variety ofsolvents were used for the nucleophilic substitution step, such asdichloromethane, chloroform and tetrahydrofuran.

In still yet another embodiment of the present invention catalyticamount of BU₄N⁺I⁻ (0.2-0.5 eq) was used by stirring the reaction mixturebetween −10° C. to room temperature for 2 to 10 h.

In still another embodiment of the present invention bases like K₂CO₃,Et₃N were also used.

In still another embodiment of the present invention the purification ofthese analogues was done by column chromatography employingchloroform/methanol as eluent.

Thus, the present invention provides new classes of podophyllotoxinanalogues, which were synthesized in a stereo selective manner.

A program was initiated in the laboratory for the design and synthesisof new 4β-aryl amino substituted podophyllotoxin congeners with enhancedantitumour activity and/or activity against etoposide resistant tumorcell lines. In these efforts new 4β-1″-[{2″-benzoylsubstituted}anilino]podophyllotoxin derivatives have been synthesizedand evaluated for their cytotoxicity and anticancer potency.Interestingly, some of the compounds have shown greater in vitrocytotoxicity values compared to etoposide. The synthesis of thesecompounds has been carried out as described in the scheme usingpodophyllotoxin obtained from the resin. The cytotoxicity of 4a-4pvalues have been illustrated in the Table 1. TABLE 1 Cytotoxicity (invitro) data for some representative compounds.

S. No. R R₁ R₂ GI₅₀ μM 4a CH₃ H H   0.04-0.5 4b H H H  15-382 4c CH₃2-Cl 4-Cl   0.059-0.876 4d H 2-Cl 4-Cl   0.1-0.24 4e CH₃ H 4-NO₂ <10nM-0.28 4f H H 4-NO₂   0.01-0.24 4g CH₃ H 4-Cl   0.07-1.1 4h H H 4-Cl 14-270 4i CH₃ 2-F 4-Cl   0.14-0.3 4j H 2-F 4-Cl   0.004-0.1 4k CH₃ H H  0.1-1 4l H H H   2-16 4m CH₃ H 4-NO₂   0.01-0.2 4n H H 4-NO₂  0.01-0.24 4o CH₃ H 4-NH₂   0.04-1 4p H H 4-NH₂   0.015-0.4

Some of the compounds of the present invention are given below:

-   -   a) 4β-1″-[2″-(Benzoyl)anilino]-4-desoxypodophyllotoxin    -   b)        4′-O-Demethyl-4β-1″-[2″-(benzoyl)anilino]-4-desoxypodophyllotoxin    -   c)        4β-1″-[2″-(2-Chlorobenzoyl)-4″-chloroanilino]-4-desoxypodophyllotoxin    -   d)        4′-O-1″-[2″-(2-Chlorobenzoyl)-4″-chloroanilino]-4-desoxypodophyllotoxin    -   e) 4β-1″-[2″-(Benzoyl)-4″-nitroanilino]-4-desoxypodophyllotoxin    -   f)        4′-O-Demethyl-4β-1″-[2″-(Benzoyl)]-4″-nitroanilino]-4-desoxypodophyllotoxin    -   g) 4β-1″-[2″-(Benzoyl)-4″-chloroanilino]-4-desoxypodophyllotoxin    -   h)        4′-O-Demethyl-4β-1″-[2″-(Benzoyl)]-4″-chloroanilino]-4-desoxypodophyllotoxin    -   i)        4β-1″-[2″-(2-Fluorobenzoyl)-4″-chloroanilino]-4-desoxypodophyllotoxin    -   j)        4′-O-Demethyl-4β-1″-[2″-(2-Fluorobenzoyl)-4″-chloroanilino]-4-desoxypodophylltoxin    -   k) 4β-1″-[3″-(Benzoyl)anilino]-4-desoxypodophyllotoxin    -   l)        4′-O-Demethyl-4β-1″-[3″-(benzoyl)anilino]-4-desoxypodophyllotoxin    -   m) 4β-1″-[2″-(Benzoyl)-2 ″-nitroanilino]-4-desoxypodophyllotoxin    -   n)        4′-O-Demethyl-4β-1″-[4″-(Benzoyl)]-2″-nitroanilino]-4-desoxypodophyllotoxin.

The following examples are given by way of illustration and should notbe construed the limit and the scope of the invention.

Experimental

EXAMPLE 1 4β-1″-[2″-(Benzoyl)anilino]-4-desoxypodophyllotoxin (4a)

4β-Bromo-4-desoxy-podophyllotoxin (0.1 g, 0.21 mmol) was reacted with2-aminobenzophenone (0.045 g, 0.23 mmol) in presence of Et₃N (0.032 g,0.32 mmol) and Bu₄N⁺I⁻ (0.015 g, 0.042 mmol) in dry tetrahydrofuran atroom temperature for 4 h. After completion of reaction solvent wasremoved in vacuo. The residue was subjected to silica gel columnchromatography using chloroform-methanol (9.8:0.2) as eluent.

Yield 60%, mp 140° C.; [α]²⁵ _(D) −112 (c, 0.1 CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 8.82 (d, 1H), 7.50 (m, 7H), 6.80 (s, 1H),6.75 (d, 1H), 6.68 (d, 1H), 6.55 (s, 1H), 6.35 (s, 2H), 5.96 (d, 2H),5.38 (s, 1H), 4.92 (m, 1H), 4.65 (d, 1H), 4.35 (t, 1H), 3.96 (t, 1H),3.82 (d, 9H), 3.20 (q, 1H), 3.50 (m, 1H)

MS (m/e) 593 (M⁺, 40%), 576, 467, 397, 282, 229, 185.

IR (KBr) cm⁻¹: 3400 (N—H), 2900 (aliphatic C—H), 1780 (lactone), 1650(ketone), 1500, 1480, 1410, 1300, 1250 (aromatic C═C).

EXAMPLE 24′-O-Demethyl-4β-1″-[2″-(benzoyl)anilino]-4-desoxypodophyllotoxin (4b)

4β-Bromo-4′-O-demethyl-4-desoxypodophyllotixin (0.1 g, 0.21 mmol) wasreacted with 2-aminobenzophenone (0.045 g, 0.23 mmol) in presence ofEt₃N (0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g, 0.042 mmol) in drytetrahydrofuran at room temperature for 4 h. After the completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.8:0.2) aseluent.

Yield 50% m.p 154-156° C.; [α]²⁵ _(D) 111 (c, 1.1, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 8.85 (d, 1H), 7.50 (m, 7H), 6.80 (s, 1H),6.75 (d, 1H), 6.68 (d, 1H), 6.55 (s, 1H), 6.35 (s, 2H), 5.96 (d, 2H),5.38 (s, 1H), 4.92 (m, 1H), 4.65 (d, 1H), 4.35 (t, 1H), 3.96 (t, 1H),3.82 (s, 6H), 3.20 (q, 1H), 3.05 (m, 1H)

MS (m/e) 579(M⁺, 25%), 495, 467, 397, 229, 185.

IR (KBr) cm⁻¹: 3550 (O—H), 3400(N—H) 2900 (aliphatic C—H), 1750(lactone), 1650 (ketone), 1500, 1480, 1410, 1300, 1250 (aromatic C═C).

EXAMPLE 34β-1″-[2″-(2-Chlorobenzoyl)-4″-chloroanilino]-4-desoxypodophyllotoxin(4c)

4β-bromo-4-desoxypodophyllotoxin (0.10 g 0.21 mmol) was reacted with2-amino-2′, 5′-dichlorobenzophenone (0.06 g, 0.23 mmol) in presence ofEt₃N (0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g 0.042 mmol) in drytetrahydrofiuran at room temperature for 5 h. After the completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.7:0.3) aseluent.

Yield 64% m.p 142-145° C.; [α]²⁵ _(D) −84 (c, 0.87, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 9.10 (d, 1H), 7.40 (m, 5H), 7.20 (d, 1H),6.78 (s, 1H), 6.75 (d, 1H), 6.52 (s, 1H), 6.35 (s, 2H), 5.96 (d, 2H),4.97 (m, 1H), 4.65 (d, 1H), 4.35 (t, 1H), 3.90 (t, 1H), 3.77 (d, 9H),3.20 (q, 1H), 3.10 (m, 1H)

MS (m/e) 663 (M⁺, 20%), 662, 661, 460, 387, 289.

IR (KBr) cm⁻¹: 3350 (N—H), 2900 (aliphatic C—H), 1760 (lactone), 1640(ketone), 1550, 1480, 1250 (aromatic C═C).

EXAMPLE 44′-O-Demethyl-4β-1″-[2″-(2-Chlorobenzoyl)-4″-chloroanilino]-4-desoxypodo-phyllotoxin(4d)

4β-Bromo-4′-O-Demethyl-4-desoxypodophyllotoxin (0.10 g 0.21 mmol) wasreacted with 2-amino-2′, 5′-dichlorobenzophenone (0.06 g, 0.23 mmol) inpresence of Et₃N (0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g 0.042 mmol)in tetrahydrofuran at room temperature for 5 h. After the completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.7:0.3) aseluent.

Yield 70% m.p 151-153° C.; [α]²⁵ _(D) −91 (c, 0.93., CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 9.10 (d, 1H), 7.40 (m, 5H), 6.77 (s, 1H),6.70 (d, 1H), 6.30 (s, 1H), 5.96 (d, 2H), 5.40 (s, 2H), 4.90 (m, 1H),4.65 (d, 1H), 4.30 (t, 1H), 4.10 (t, 1H), 3.80 (s, 6H), 3.20 (q, 1H),3.10 (m, 1H).

MS (m/e) 649 (M⁺, 20%), 648, 647, 446, 383, 289.

IR (KBr) cm⁻¹: 3320 (N—H), 2900 (aliphatic C—H), 1760 (lactone), 1650(ketone), 1550, 1480, 1410, 1250 (aromatic C═C).

EXAMPLE 5 4β-1″-[2″-(Benzoyl)-4″-nitroanilino]-4-desocypodophyllotocin(4e)

4β-Bromo-4-desoxypodophyllotoxin (0.1 g, 0.21 mmol) was reacted with2-amino-5-nitro-benzophenone (0.056g, 0.23 mmol) in presence of Et₃N(0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g, 0.042 mmol) in drytetrahydrofuran at room temperature for 8 h. After completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.5:0.5) aseluent.

Yield 40%, mp 163-167° C.; [α]²⁵ _(D) −85 (c, 1.2, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 9.50 (d, 1H), 8.57 (d, 1H), 8.32 (q, 1H),7.60 (m, 4H), 6.75 d, 1H), 6.75 (s, 1H) 6.60 (d, 1H), 6.40 (d, 1H), 6.30(s, 2H), 6.00 (d, 2H), 5.05 (m, 1H), 4.70 (d, 1H), 4.40 (t, 1H), 3.90(t, 1H) 3.80 (d, 9H), 3.15 (d, 1H), 2.95 (m, 1H)

MS (m/e) 638 (M⁺, 10%), 582, 496, 439, 411, 383, 289.

IR (KBr) cm⁻¹: 3450 (N—H), 2950 (aliphatic C—H), 1740 (lactone), 1650(ketone), 1550, 1480, 1250 (aromatic C═C).

EXAMPLE 64′-O-Demethyl-4β-1″-[2″-(Benzoyl)]-4″-nitroanilino]-4-desoxypodophyllotoxin(4f)

4β-Bromo-4′-O-demethyl-4-desoxypodophyllotoxin (0.1 g, 0.21 mmol) wasreacted with 2-amino-5-nitro-benzophenone (0.056 g, 0.23 mmol) inpresence of Et₃N (0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.15 g, 0.042 mmol)in dry tetrahydrofuran at room temperature for 8 h. After completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.5:0.5) aseluent.

Yield 38%, mp 169-171° C.; [α]²⁵ _(D) −89 (c, 1.0, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 9.47 (d, 1H), 8.55 (d, 1H), 8.30 (q, 1H),7.60 (m, 4H), 6.80 (d, 1H), 6.55 (s, 1H) 6.35 (d, 1H), 6.30 (s, 2H),6.00 (d, 2H), 5.87 (s, 1H), 5.00 (m, 1H), 4.65 (d, 1H), 4.30 (m, 2H),3.80 (d, 6H), 3.15 (d, 1H), 2.00 (m, 1H)

MS (m/e) 624(M⁺, 15%), 568, 401, 383,289, 229, 185.

IR (KBr) cm⁻¹: 3560 (O—H), 3400 (N—H) 2900 (aliphatic C—H), 1740(lactone), 1650 (ketone), 1500, 1480, 1250 (aromatic C═C).

EXAMPLE 7 4β-1″-[2″-(Benzoyl)-4″-chloroanilino]-4-desoxypodophyllotoxin(4g)

4β-bromo-4-desoxypodophyllotoxin (0.10 g 0.21 mmol) was reacted with2-amino-5-chlorobenzophenone (0.053 g, 0.23 mmol) in presence of Et₃N(0.032 g, 0.32 mmol) and Bu₄N⁺F⁻ (0.015 g 0.042 mmol) in at roomtetrahydrofuran temperature for 6 h. After the completion of reactionsolvent was removed in vacuo. The residue was subjected to silica gelcolumn chromatography using chloroform-methanol (9.7:0.3) as eluent.

Yield 56% m.p 139-142° C.; [α]²⁵ _(D) −103 (c, 0.93, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 8.72 (d, 1H), 7.60 (m, 7H), 7.45 (q, 1H),6.80 (s, 1H), 6.75 (d, 1H), 6.55 (s, 1H), 6.35 (s, 2H), 5.98 (d, 2H),4.95 (m, 1H), 4.65 (d, 1H), 4.40 (t, 1H), 3.95 (t, 1H), 3.80 (d, 9H),3.20 (q, 1H), 3.10 (m, 1H)

MS (m/e) 628 (M⁺, 20%), 627, 441, 383, 289, 229, 185

IR (KBr) cm⁻¹: 3350 (N—H), 2900 (aliphatic C—H), 1780 (lactone), 1660(ketone), 1500, 1480, 1410, 1250 (aromatic C═C).

EXAMPLE 84′-O-Demethyl-4β-1″-[2″-(Benzoyl)]-4″-chloroanilino]-4-desoxypodophyllotoxin(4h)

4β-Bromo-4′-O-demethyl-4-desoxypodophyllotoxin (0.1 g, 0.21 mmol) wasreacted with 2-amino-5-chlorobenzophenone (0.053 g, 0.23 mmol) inpresence of Et₃N (0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g, 0.042 mmol)in dry tetrahydrofuran at room temperature for 6 h. After completion ofthe reaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.7:0.3) aseluent.

Yield 50%, mp 146-149° C.; [α]²⁵ _(D) −105 (c, 0.97, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 8.68 (d, 1H), 7.52 (m, 7H), 7.35 (q, 1H),6.72 (s, 1H), 6.65 (d, 1H), 6.50 (s, 1H) 6.30 (s, 2H), 5.96 (d, 2H),5.35 (s, 1H), 4.85 (m, 1H), 4.60 (d, 1H), 4.30 (t, 1H), 3.85 (d, 1H),3.80 (s, 6H), 3.10 (q, 1H), 3.00 (m, 1H)

MS (m/e) 614 (M⁺, 10%), 613, 401, 383, 289, 229, 185

IR (KBr) cm⁻¹: 3500 (O—H), 3360 (N—H) 2900 (aliphatic C—H), 1750(lactone), 1640 (ketone), 1500, 1480, 1230 (aromatic C═C).

EXAMPLE 94β-1″-[2″-(2-Fluorobenzoyl)-4″-chloroanilino]-4-desoxypodophyllotoxin(4i)

4β-bromo-4-desoxypodophyllotoxin (0.10 g 0.21 mmol) was reacted with2-amino-5-chloro-2′-fluorobenzophenone (0.057 g, 0.23 mmol) in presenceof Et₃N (0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g 0.042 mmol) in drytetrahydrofuran at room temperature for 5 h. After the completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.7:0.3) aseluent.

Yield 68% m.p 123-128° C.; [α]²⁵ _(D) −89 (c, 1.0, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 9.10 (d, 1H), 7.45 (m, 6H), 6.80 (s, 1H),6.75 (d, 1H), 6.55 (s, 1H), 6.35 (s, 2H), 6.00 (d, 2H), 4.95. (m, 1H),4.70 (d, 1H), 4.40 (t, 1H), 3.95 (t, 1H), 3.82 (d,9H), 3.10(m,1H)

MS (m/e) 646 (M⁺, 30%), 645, 631, 411, 397, 229, 185

IR (KBr) cm⁻¹: 3400 (N—H), 2950 (aliphatic C—H), 1760 (lactone), 1650(ketone), 1500, 1480, 1300, 1250 (aromatic C═C).

EXAMPLE 104′-O-Demethyl-4β-1″-[2″-(2-Fluorobenzoyl)-4″-chloroanilino]-4-desoxypodo-phyllotoxin(4j)

4β-Bromo-4′-O-Demethyl-4-desoxypodophyllotoxin (0.10 g 0.21 mmol) wasreacted with 2-amino-5-chloro-2-fluorobenzophenone (0.057 g, 0.23 mmol)in presence of Et₃N (0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g 0.042mmol) in dry tetrahydrofuran at room temperature for 5 h. After thecompletion of reaction solvent was removed in vacuo. The residue wassubjected to silica gel column chromatography using chloroform-methanol(9.7:0.3) as eluent.

Yield 60% m.p 164-167° C.; [α]²⁵ _(D) −85 (c, 1.01, CHCl₃) ¹H NMR (200MHz, CDCl₃): δ 9.05 (d, 1H), 7.48 (m, 6H), 6.80 (s, 1H), 6.75 (d, 1H),6.52 (s, 1H C-8), 6.35 (s, H), 6.00 (d, 2H), 5.10 (s, 1H), 4.98 (m, 1H),4.70 (t, 1H), 4.40 (t, 1H), 3.95 (t, 1H), 3.82 (s, 6H), 3.20 (q, 1H),3.10 (m, 1H).

MS (m/e) 632 (M⁺, 25%), 631, 401, 383, 229, 185

IR (KBr) cm⁻¹: 3520 (O—H), 3440(N—H), 2900 (aliphatic C—H), 1750(lactone), 1650 (ketone), 1500, 1480, 1300, 1250 (aromatic C═C).

EXAMPLE 11 4β-1″-[3″-(Benzoyl)anilino]-4-desoxypodophyllotoxin (4k)

4β-Bromo-4-desoxypodophyllotoxin (0.1 g, 0.21 mmol) was reacted with2-amino-4-bromobenzo-phenone (0.064 g, 0.23 mmol) in presence of Et₃N(0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g, 0.042 mmol) in drytetrahydrofuran at room temperature for 3 h. After completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.8:0.2) aseluent.

Yield 61%, mp 141-144° C.; [α]²⁵ _(D) −102 (c, 1.1, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 7.80 (d, 1H), 7.50 (m, 3H), 7.25 (d, 1H),7.10 (d, 1H), 7.01(s, 1H), 6.80 (s, 1H), 6.75 (d, 1H), 6.50 (s, 1H),6.28 (s, 2H), 5.95 (d, 2H), 5.30 (s, 1H), 4.75 (m, 1H), 4.55 (d, 1H),4.40 (t, 1H), 4.00 (t, 1H) 3.75 (q, 9H), 3.05 (m, 2H)

MS (m/e) 593 (M⁺25%), 576, 467, 397, 229, 185.

IR (KBr) cm⁻¹: 3400 (N—H), 2950 (aliphatic C—H), 1760 (lactone), 1650(ketone), 1500, 1480, 1250 (aromatic C═C).

EXAMPLE 124′-O-Demethyl-4β-1″-[3″-(benzoyl)anilino]-4-desoxypodophyllotoxin (4l)

4β-Bromo-4′-O-demethyl-4-desoxypodophyllotixin (0.1 g, 0.21 mmol) wasreacted with 3-aminobenzophenone (0.045 g, 0.23) in presence of Et₃N(0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g, 0.042 mmol) in drytetrahydrofuran at room temperature for 3 h. After the completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.8:0.2) aseluent.

Yield 63% m.p 151-154° C.; [α]²⁵ _(D) −102; (c, 1.1. CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 7.84 (d, 2H), 7.52 (m, 3H), 7.25 (d, 1H),7.10 (d, 1H), 7.01(s, 1H), 6.90 (s, 1H), 6.78 (d, 1H), 6.54 (s, 1H),5.95 (d, 2H), 4.75 (m, 1H), 4.55 (d, 1H), 4.40 (t, 1H), 4.00 (t, 1H),3.75 (d, 9H), 3.05 (m, 2H)

MS (m/e) 579 (M⁺, 25%) 382, 283, 229, 185.

IR (KBr) cm⁻¹: 3520 (O—H), 3390 (N—H) 2900 (aliphatic C—H), 1760(lactone), 1650 (ketone), 1500, 1480, 1250 (aromatic C═C).

EXAMPLE 13 4β-1″-[2″-(Benzoyl)-2″-nitroanilino]-4-desoxypodophyllotoxin(4m)

4β-Bromo-4-desoxypodophyllotoxin (0.1 g, 0.21 mmol) was reacted with4-amino-3-nitro-benzophenone (0.056 g, 0.23 mmol) in presence of Et₃N(0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g, 0.042 mmol) in drytetrahydrofuran at room temperature for 8 h. After completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.4:0.6) aseluent.

Yield 42%, mp 163-167° C.; [α]²⁵ _(D) −81 (c, 0.9, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 8.68 (d, 1H), 8.50 (d, 1H), 8.05 (d, 1H),7.75 (d, 2H), 7.55 (m, 3H), 6.93 (d, 1H) 6.750 (s, 1H), 6.58 (s, 1H)6.50 (d, 1H), 6.32 (s, 2H), 6.00 (d, 2H), 5.32 (d, 1H), 5.07 (m, 1H),4.68 (d, 1H), 4.35 (t, 1H), 3.92 (t, 1H), 3.80 (d, 9H), 3.13 (d, 1H) MS(m/e) 638 (M⁺, 15%), 582, 524, 428, 411, 383, 289.

IR (KBr) cm⁻¹: 3400 (N—H), 2950 (aliphatic C—H), 1760 (lactone), 1640(ketone), 1500, 1480, 1410, 1300, 1250 (aromatic C═C).

EXAMPLE 144′-O-Demethyl-4β-1″-[4″-(Benzoyl)]-2″-nitroanilino]-4-desoxypodophyllotoxin(4n)

4β-Bromo-4′-O-demethyl-4-desoxypodophyllotoxin (0.1 g, 0.21 mmol) wasreacted with 4-amino-3-nitrobenzophenone (0.056 g, 0.23 mmol) inpresence of Et₃N (0.032 g, 0.32 mmol) and Bu₄N⁺I⁻ (0.015 g, 0.042 mmol)in dry tetrahydrofuran at room temperature for 8 h. After completion ofreaction solvent was removed in vacuo. The residue was subjected tosilica gel column chromatography using chloroform-methanol (9.6:0.4) aseluent.

Yield 34%, mp 170-175° C.; [α]²⁵ _(D) −85 (c, 1.0, CHCl₃)

¹H NMR (200 MHz, CDCl₃): δ 8.70 (d, 1H), 8.50 (d, 1H), 8.10 (d, 1H),7.75 (m, 3H), 6.93 (d, 1H), 6.72 (s, 1H) 6.58 (s, 1H), 6.32 (s, 2H),6.00 (d, 2H), 5.40 (s, 1H), 5.05 (m, 1H), 4.65 (d, 1H), 4.35 (t, 2H),3.90 (t, 1H), 3.80 (s, 6H), 3.10 (d, 2H)

MS (m/e) 624 (M⁺, 15%), 467, 401, 229, 185

IR (KBr) cm⁻¹: 3530 (O—H), 3450 (N—H) 2900 (aliphatic C—H), 1750(lactone), 1650 (Ketone), 1500, 1480, 1410, 1250 (aromatic C═C).

In conclusion, the main advantages of the present inventions are thatthese new 4β-1″-[2″-(substituted benzoyl)anilino] podophylotoxinanalogues have exhibited promising in vitro cytotoxic activity andenhanced potential as anticancer agents. Further, these compounds havebeen prepared 4β-bromopodophylltoxin upon reaction with thecorresponding 2-aminobenzophenone in the presence of Et₃N and BU₄N⁺I⁻ atroom temperature to provide the 4β-1″-[2″-(substitutedbenzoyl)anilino]podophylotoxin analogues in very good yields and inalmost stereoselective manner.

1. New analogues of 4β-1″-[{2″-benzoylsubstituted)anilino]podophyllotoxin of FIG. 2 having anticancer activitywherein R, R₁ and R₂ independently or in combination represents R═H orCH₃ R₁═H or halogen R₂═H, NO₂ or halogen
 2. Analogues of4β-1″-[{2″-benzoyl substituted}anilino]podophyllotoxin as claimed inclaim 1 wherein the preferred compounds and their cytotoxicity valuesare as follows: S. No R R₁ R₂ GI₅₀ μ M 4a CH₃ H H 0.04-0.5  4b H H H 15-382 4c CH₃ 2-Cl 4-Cl 0.059-0.876 4d H 2-Cl 4-Cl  0.1-0.24 4e CH₃ H4-NO₂ <10 nM-0.28   4f H H 4-NO₂ 0.01-0.24 4g CH₃ H 4-Cl 0.07-1.1  4h HH 4-Cl  14-270 4i CH₃ 2-F 4-Cl 0.14-0.3  4j H 2-F 4-Cl 0.004-0.1  4k CH₃H H 0.1-1   4l H H H  2-16 4m CH₃ H 4-NO₂ 0.01-0.2  4n H H 4-NO₂0.01-0.24 4o CH₃ H 4-NH₂ 0.04-1   4p H H 4-NH₂ 0.015-0.4 


3. Few analogues of 4β-1″-[{2″-benzoylsubstituted)anilino]podophyllotoxin as claimed in claim 2 have better invitro anti-cancer activity compared to the standard anti-cancer drugEtoposide.
 4. A process for the preparation of 4β-1″-[{2″-benzoylsubstituted}anilino]podophyllotoxin anolgues of FIG. 2 wherein R, R₁ andR₂ independently or in combination represents R═H or CH₃ R₁═H or halogenR₂═H, NO₂ or halogen the said process comprising the following steps: a)reacting 4β-bromo-4-dioxypodophyllotoxin with substituted orunsubstituted 2-aminobenzophenone in presence of phase transfercatalyst, base in an anhydrous organic solvent medium at a temperatureranging between −10° to 40° C. for 4-16 hrs. b) removing the organicsolvent from the reaction mixture of step (a) under reduced pressure toobtain a residue, and c) purifying the residue of step (b) over silicagel column, eluting with mixture of chloroform-methanol to obtain therequired 4β-1″-[{2″-benzoyl substituted}anilino]podophyllotoxinanalogues.
 5. A process as claimed in claim 3 wherein in step (a), thephase transfer catalyst used is selected from a group consisting oftetrabutyl ammonium chloride, tetrabutyl ammonium bromide, tetra butylammonium iodide or aliquat 336 and preferably tetrabutylammonium iodide.6. A process as claimed in claim 3 wherein in step (a), the substituted2-amino benzophenone are selected from group consisting of2-amino-2′,5′-dichlorobenzophenone, 2-amino-5-nitrobenzophenone,2-amino-5-chlorobenzophenone, 2-amino-5-chlorobenzophenone,2-amino-5-chloro-2′-fluorobenzophenone, 2-amino-4′bromobenzophenone, or4-amino-3-nitrobenzophenone.
 7. A process as claimed in claim 3 whereinin step (a), the organic solvent used is selected from group consistingof dichlorormethane, chloroform, tetrahydrofuran or dioxane andpreferably tetrahydrofuran.
 8. A process as claimed in claim 3 whereinin step (a), the base used is selected from group consisting oftrimethylamine, triethylamine, sodium carbonate, potassium carbonate,cesium carbonate or barium carbonate and preferably triethylamine.
 9. Aprocess as claimed in claim 3 wherein the reaction is preferably carriedout at room temperature.
 10. A process as claimed in claim 3 wherein instep (a), the molar ratio of substituted or unsubstituted benzophenoneand the bromocompound used is in the range of 1:1 to 2:1 and preferably1:1.17
 11. A process as claimed in claim 3 wherein in step (a), the moleequivalent ratio of bromo compound to phase transfer catalyst is in therange of 1:0.2 to 1:0.5.
 12. Use of 4β-1″-[{2″-benzoylsubstituted}anilino]podophyllotoxin analogues of general formula (2) asclaimed in claims 1 and 2 for treating cancer in a subject comprisingadministering a pharmaceutically effective dosage of the said analoguesoptionally with additives to the said subject in need thereof.
 13. Useas claimed in claim 12 wherein 4β-1″-[{2″-benzoylsubstituted}anilino]podophyllotoxin analogues are used singly or incombination with pharmaceutically acceptable carriers.
 14. Use asclaimed in claim 12 wherein the analogues of general formula (2) areadministered systemically or orally.
 15. Use as claimed in claim 12wherein the subject is selected from mammals.
 16. Use as claimed inclaim 15 wherein the subject excludes humans.
 17. Use as claimed inclaim 12, wherein the compounds of general formula (2) is administeredto the subject in combination with pharmaceutically acceptableadditives, carriers, diluent, solvent, filter, lubricant, excipient,binder or stabilizer.
 18. Use as claimed in claim 12 wherein the GI 50value of in vitro anti-cancer activity of preferred analogues is in therange of 0.001-382 μM